Zinc-enriched biomass, method for the preparation thereof and pro-biotic, cosmetic, dietary and nutraceutic products comprising the same

ABSTRACT

The invention relates to a zinc-enriched biomass comprising living or subsequently killed micro-organisms, selected from the group consisting of  Streptococcus thermophilus  and  Bifidobacterium animalis  subsp.  lactis  and combinations thereof, a method for the manufacture of said zinc-enriched biomass, as well as food preparations, nutraceutic products, functional foods, cosmetic and cosmeceutic products, and food supplements, comprising the said biomass. Furthermore, new micro-organism strains are described which are able to concentrate zinc within the cell in very high amounts and therefore are particularly suitable for use in the method of the invention.

The present invention relates to a zinc-enriched biomass, a method forthe preparation thereof, as well as foodstuffs, pro-biotic, dietary,nutraceutic and cosmetic products comprising the same. The inventionfurther relates to bacterial strains suitable to be used in the methodof the invention.

Zinc is an essential mineral that is present in organisms in amountshigher than those of any other oligoelement, with the exception of iron.It is linked to the normal absorption of vitamins and their activity,particularly the B-complex vitamins. It is a constituent element of ahuge number of enzymes that play a role in digestion and metabolism,including carbonic anhydrase, required for tissue respiration. In thehuman body, zinc is especially found in bones, teeth, skin, liver,muscles and hair. Zinc is rapidly absorbed into the higher portion ofthe small intestine. Zinc also deposits itself in certain eyestructures, prostate, spermatozoa, skin, hair, nails and is found inwhite blood cells too. These supplies are not easily usable, thereforethe food has to contain sufficient quantities of it in order to meet theorganism's requirements. It is indispensable for body growth, tissuerepair, and for a normal immune response. It is also important forcarbohydrate digestion and phosphorous metabolism. It participates inthe synthesis of nucleic acid that controls the creation of variousproteins in cells, it is important for vitamin absorption, it is usefulin healing processes, and it inhibits the bacterial, yeast, and skinsaprophyte lipases. Very many enzymes need zinc to be active, which isnecessary for protein synthesis, for certain aspects of hormonalfunctions, for brain functions, sight, and taste. Furthermore, thealcohol dehydrogenase enzyme—involved in alcohol breakdown—containszinc, therefore alcohol causes a loss of zinc. Zinc is used to reducesebaceous secretions, in healing processes for internal and externalwounds (it accelerates the healing of wounds), in therapies for acne andseborrhoeic dermatitis. This metal can promote hair re-growth in peoplewho suffer from alopecia aerata totalis and be used in diabetes therapy,thanks to its regulatory effects on blood insulin. Adding zinc toinsulin has been found to extend the effect of the hormone on bloodsugar levels.

Zinc-deficiency causes serious disorders in all living beings. It isknown that certain drugs can induce a zinc-deficiency, among which theanti-MAOs (anti-Monoamine oxidase), corticosteroids, diuretics.Zinc-deficiency can cause growth retardation, delayed sexual maturation,and longer times for wound-healing. Zinc-deficiency can also lead toatherosclerosis and increase susceptibility to infections. Stretch marksand nail white spots can be symptoms of zinc-deficiency. Other symptomsof zinc-deficiency are fragile nails and hair, lack of hair pigment,irregular menstrual cycles in adolescent girls, impotence in youngmales, and knee and hip joint pains in adolescents. Chroniczinc-depletion can even pre-dispose body cells to cancer. Even smallzinc-deficiencies are deleterious to the organism, for instance they candetermine a reduction in the concentration of spermatozoa and impotence.Furthermore, zinc-deficiency causes fatigue, higher chances ofcontracting infections or experiencing wounds, and reduced mentalagility. In fact, zinc-deficiency hinders energy production, proteinsynthesis, collagen formation, and alcohol tolerance.

Food or dietary compositions containing zinc in combination withpro-biotic agents are described in the prior art.

For example, US patent application 20070009502 A describes nutritionalcompositions for animal feed, designed for the improvement ormaintenance of the gastrointestinal microflora, comprising pro-bioticagents (such as yeast and/or bacteria, for example Bifidobacterium,Enterococcus or Lactobacillus), pre-biotic agents, glutamine or itsanalogues, glucose, glycine, electrolytes, vitamins and minerals,including mineral zinc (100-200 mg/kg).

Patent application WO 2006/112998 describes a liquid nutritionalsupplement to be used in combination with human milk, designed topromote the growth of breast-fed babies suffering growth retardation,comprising numerous ingredients, among which pro-biotics (such asLactobacillus and/or Bifidobacterium) and minerals, including zinc.

Patent application CA 2525342 A describes a broad-range pro-biotic foodpreparation useful in food supplements, for instance for improving theimmune response against diseases, comprising specific bacterial strainsof Lactobacillus plantarum, Lactobacillus fermentum, Lactobacillus caseisbp. pseudoplantarum, Lactobacillus acidophilus and Lactobacillus casei,in combination with further auxiliary components, linkers and energizingcomponents, including zinc.

The above-mentioned prior documents describe compositions suitable forzinc delivery within the human or animal body, wherein zinc is in theform of inorganic zinc and is in combination with many otheringredients, including pro-biotic micro-organisms.

These compositions have the disadvantage that they contain zincexclusively in the inorganic form, which is more difficult for the humanbody to absorb than organic zinc.

The inventors have now found that certain bacterial species belonging tothe Bifidobacterium and Streptococcus genera, especially theBifidobacterium animalis and Streptococcus thermophilus species, whengrown on a culture medium containing inorganic zinc, display theunexpected and advantageous ability to accumulate extremely highquantities of zinc within the cell, without such high amounts ofintracellular zinc which is deleterious for the survival of the biomassitself. Such a capacity to accumulate zinc intracellularly makes theaforesaid bacterial species particularly suitable for use as a zincdelivery means within the human or animal body, particularly useful forthe manufacture of pro-biotic products that, by definition, must containa living biomass. The zinc-enriched biomass of the invention may also beused in cosmetic applications, especially for the manufacture ofcosmetic or cosmeceutic products. For the manufacture of cosmeticproducts, the biomass needs to be made up of dead micro-organisms,whereas for the manufacture of cosmeceutic products, the biomass needsto be made up of living micro-organisms.

Thus, one object of the invention is a method for the manufacture of azinc-enriched biomass, characterised in that the biomass is obtained by

-   -   (i) culturing micro-organisms selected from the group consisting        of Bifidobacterium animalis, Streptococcus thermophilus, and        combinations thereof, in a nutrient culture medium comprising a        zinc salt, so that the said micro-organisms accumulate zinc at        an intracellular level; and    -   (ii) separating the zinc-enriched micro-organisms from the        culture medium.

A biomass comprising living micro-organisms that contain a high quantityof zinc accumulated within the cells is achieved by the method of theinvention, as is evident from the studies reported hereinafter.

The method for the manufacture of the zinc-enriched biomass of theinvention provides for a first fermentation step, wherein themicro-organisms are cultured in a nutrient medium suitable for growingmicro-organisms from the Bifidobacterium and Streptococcus generasupplemented with a zinc salt, preferably zinc sulphate (ZnSO₄). Thenutrient medium is preferably a liquid medium containing carbon sources,for instance glucose and/or lactose; nitrogen sources, for examplepeptones, casein hydrolysates, yeast extracts; inorganic salts;micro-elements and vitamin sources.

The concentration of the zinc salt in the culture medium is preferablybetween 5 and 50 mM, even more preferably between 10 and 40 mM.

Zinc sulphate is preferred.

The fermentation is preferably carried out at a temperature between 25°C. and 48° C., more preferably between 35° C. and 45° C. The pH value ofthe liquid medium preferably is between 2.5 and 8.0, more preferablybetween 3.5 and 7.5. The fermentation time length is preferably between6 and 40 hours, more preferably between 8 and 36 hours. The fermentationmay be carried out under aerobic, micro-aerobic, and/or anaerobicconditions.

Following the fermentation step, during which biomass growth and zincaccumulation within the bacterial cells occur, the biomass obtained isseparated from the culture medium by any suitable per se known method,for example by centrifugation or micro-filtration, in such a way as notto compromise the cell viability. Thus, the method of the inventionallows a zinc-enriched micro-organism biomass to be obtained comprisingliving micro-organisms. If desired, the biomass obtained may then besubjected to freeze-drying, drying, micro-encapsulation and/or freezing,carried out according to conventional procedures.

The present inventors have also selected two micro-organism strains fromthe Streptococcus thermophilus and Bifidobacterium animalis species,which proved to be particularly advantageous for use in the method ofthe invention, as they are endowed with a particularly high ability toaccumulate zinc within the cell. Such strains have been designated asStreptococcus thermophilus ST 16 BM and Bifidobacterium animalis subsp.lactis BB 1 BM and have been deposited at the DSMZ (Deutsche Sammlungfür Mikroorganismen and Zellkulturen, Braunschweig, Germany), under theBudapest Treaty, as Streptococcus thermophilus ST 16 BM deposited onJul. 13, 2007, under the accession number DSM 19526, and Bifidobacteriumanimalis subsp. lactis BB 1 BM deposited on Dec. 23, 2005, under theaccession number DSM 17850, respectively.

As previously described, the zinc-enriched biomass achievable by themethod of the invention is particularly suitable for use as a pro-bioticagent, in that it contains high zinc concentrations in the organic form.

To this end, the biomass comprising living micro-organisms, andtherefore having pro-biotic activity, may be prepared in differentforms. For instance, it can be added to a food product, preferably amilk or a dairy product such as yoghurt, in order to obtain a foodpreparation having pro-biotic activity. Alternatively, it may be usedfor the manufacture of a composition having pro-biotic activity, such asfor example a food supplement, a dietary product, a functional food, orfor the manufacture of a non-food preparation for oral administration,such as for example a nutraceutic product, in combination with suitablevehicles and/or excipients. To this end, the biomass is preferably usedin the form of a freeze-dried or dried composition as such and/or of amicro-encapsulated composition.

The bacterial load of the freeze-dried or dried product to besubsequently used in the composition having pro-biotic activity is atleast of 10¹⁰-10¹¹ CFU/g of product.

For the manufacture of the freeze-dried or dried product, the wetbiomass is suspended in a liquid medium, for example water or a sterilephysiologic solution, with the inclusion of protective agents such asfor example skimmed milk, lactose, glucose, yeast extract, potatostarch, sodium glutamate, inositol, sodium citrate, gelatine,maltodextrin, magnesium stearate, ascorbic acid, stearic acid andcombinations thereof.

The freeze-dried or dried product is then diluted for the manufacture ofpro-biotics with inert substances selected for example from the onesindicated above for the freeze-drying, such as to obtain a bacterialload preferably of at least 10⁹ CFU/g of product. The freeze-driedproduct may be micro-encapsulated in order to increase the stability atroom temperature (18-24 months).

For the manufacture of a product wherein the biomass must be dead (forinstance a cosmetic product or some food products, such as bakeryproducts), the zinc-enriched biomass achievable by the method of theinvention is subjected to per se known methods, such as drying, toobtain dead cells.

The following experimental section is provided exclusively by way ofillustration and is not intended to limit the scope of the invention asdefined in the attached claims.

Tests of Intracellular Zinc Uptake

In order to compare the total intracellular zinc contents and toestimate the accumulation capacity of the metal at the cytoplasmic ormembrane level, we carried out a screening on pro-biotic micro-organismstrains grown on media with or without zinc sulphate.

Bifidobacterium cultures stored in MRS infussion were inoculated intoliquid MRS and anaerobically incubated at 37° C. After a 24 hour growthperiod, 120 ml (10% v/v) of liquid MRS alone and 120 ml (10% v/v) ofliquid MRS supplemented with 10 mM ZnSO₄, were inoculated, respectively.These cultures were incubated under anaerobic conditions at 37° C. for48 hours. The same experimental procedure was followed to set up testsof intracellular uptake in Streptococcus. In this case, a liquid M17medium was used and the cultures were incubated under anaerobicconditions at 42° C. for 48 hours.

At the end of the growth, having kept a small aliquot for thedetermination of the dry weight, we proceeded to separate the biomassfrom the culture medium by centrifugation and to mineralize thecollected biomass.

Biomass Mineralization

In order to determine the total intracellular zinc, the bacterial cellswere completely disrupted and then the biomass was mineralized accordingto the protocol reported hereinafter.

One hundred ml of the grown cultures were centrifuged for 30 minutes at4500 rpm (in a centrifuge cooled down to 4° C., Beckman GS-15Rcentrifuge) to collect the cells. The pellet was then washed 4 times,each time with 140 ml of distilled water, in order to eliminate theresidual zinc from the supernatant. The fourth water wash was retainedto analyze its zinc contents by the ICP technique.

The biomass was mineralized by re-suspending the pellet in a 1:1 ratio(w/v) with a solution of nitric acid HNO₃. During the optimization ofthe mineralization procedure, in order to achieve total recovery of theintracellular zinc, we used increasing concentrations of nitric acidsolutions, 0.65%, 6.5%, and 20%, respectively.

Each so-obtained cell suspension was transferred into screw-cap tubesand stored at −20° C. for at least 2 hours. The screw-cap tubes werethen thawed in a thermostat bath at 100° C. for 30 minutes under achemical hood: to avoid excessive evaporation and pressure within thetubes, these were sealed with stoppers fitted with a vent needle. Thesolutions were cooled at room temperature, allowing for the reactionvapours to be given off. At the end of the mineralization procedure, thecell suspensions were centrifuged at 13000 rpm for 30 minutes at 4° C.in order to collect the mineralized extract and to eliminate the celldebris.

Total Zinc Analysis by ICP

For the analysis by ICP, the samples were acidified at 2% with65%-concentrated HNO₃ and then diluted with bi-distilled water up to a 5ml final volume. These operations were effected under a chemical hood.The solutions thus obtained were filtered by using 0.8 μm celluloseacetate filters (Millex-AA, Millipore) until they were completely clear.

The quantification of the intracellular zinc accumulated by the strainsunder examination was carried out by the ICP-AES technique (OES-OPTIMA4200 DV, Perkin Elmer).

The system uses frequencies of 40 MHz. The plasma injection is automatedand controlled by a computer-connected electronic system.

The argon used must be 99.99% pure and its flow must always be in arange from 0 to 20 litres/minute, with variable increases of 1litre/minute. The nebulised sample flow must occur within mass flowvalues from 0 to 0.01 litres/minute, with variable increases of 1litre/minute.

The nebuliser is made up of corrosion-resistant materials, so the systemcan withstand solutions with 50% (v/v) concentrations of HCl, HNO₃,H₂SO₄, H₃PO₄, 30% (v/v) concentrations of NaOH and 20% (v/v)concentrations of HF.

The spectrophotometer consists of a polycromator that lies in athermostat compartment at 38° C. The detection method used is the CCDand the reading out is done in the UV field.

Analysis parameters used:Resolution: highPurge gas flow: normalRead delay time (sec): 45

Replicates: 3

Read time: autoMin. time: 1.000 sec.-Max. time: 10.000 sec.Source equilibration delay: 15 sec.Plasma aerosol type: wetNebulizer start-up conditions: instant

In order to obtain the unknown zinc concentration, we used a calibrationline constructed with the following standard solutions 2% acidified withHNO₃: 0.5 ppm, 1 ppm, 2 ppm, and 10 ppm zinc.

The total cell zinc is expressed in mg of metal per gram of dry biomass.

The total intracellular concentration of the accumulated metal wasdefined by the ICP-AES technique described above by acidifying thesamples obtained at 2% with HNO₃.

FIG. 1 reports the intracellular zinc concentrations (expressed as mg ofintracellular zinc per gram of dry cell weight) measured in uptake testscarried out on strains of different species of Bifidobacterium.Specifically, the tests were effected on strains of B. infantis, B.breve, B. bifidum, B. animalis, B. longum. All the analysed strains showa very low intracellular zinc concentration, with values lying between0.01 and 0.20 mg/g_(DW), when grown on zinc-free MRS medium. Theaddition to the MRS culture medium of 10 mM zinc sulphate induces anincrease in the intracellular zinc concentration. However, as can beseen from FIG. 1, the concentrations of the internalised metal arerather low and lie between 0.72 mg/g_(DW) and 2.12 mg/g_(DW) in thestrains of all species, except for the Bifidobacterium animalis subsp.lactis BB 1 BM strain (accession number DSM 17850; filing date Dec. 23,2005) wherein the intracellular zinc concentration increases up to 53.32mg/g_(DW) (an increase of as much as 1.000 times the basalconcentration).

As can be seen from FIG. 2, comparable intracellular zinc values havebeen achieved with the Streptococcus thermophilus ST 16 BM strain(accession number DSM 19526; filing date Jul. 13, 2007) grown on M17medium enriched with 10 mM zinc sulphate (intracellular zincconcentration=59.31 mg/g_(DW)) (Series 1). FIG. 2 further shows thatstrains belonging to other Streptococcus species, such as S. salivarius,S. faecium and S. lactis, demonstrate an much lower ability toaccumulate intracellular zinc when grown on the same medium.Furthermore, the figure shows the comparison with the intracellular zincconcentrations detected when the strains are grown on the same M17medium without zinc sulphate added (Series 2).

EXAMPLE 1

200 ml of M17 medium (Merck), to which 10 mM zinc sulphate has beenadded, were sterilised in a 500 ml flask. A seed culture ofStreptococcus thermophilus ST 16 BM (accession number DSM 19526; filingdate Jul. 13, 2007), previously grown for 24 hours at 42° C. underanaerobic conditions, was inoculated into the flask in the amount of 10%(v/v). The culture was then left growing for 40 hours at 42° C. underanaerobic conditions. 2.73×10⁹ CFU/ml were obtained at the end of theculture. The biomass was collected by centrifugation and treated andanalysed according to the methods described above. The total zincaccumulated by the cells was 59.31 mg/g_(DW). D.W.=dry weight.

EXAMPLE 2

As in Example 1, using SBF31 medium+15 mM Zn²⁺ separately sterilised byfiltration. SBF31 medium: tryptone 23 g/l; soybean peptone 16 g/l; yeastextract 12 g/l; MgSO₄ 0.25 g/l; K₂HPO₄ 2.5 g/l; ascorbic acid 0.5 g/l;glucose 45 g/l; Na diglycerophosphate 19 g/l.

Fermentation time: 21 hours; production of living cells: 5.3×10¹⁰CFU/ml; accumulated zinc: 51.67 mg/g_(DW).

EXAMPLE 3

As in Example 1, using SBF32 medium+30 mM Zn²⁺ separately sterilised byfiltration. After 21 hours, 2.1 g/litre of dry biomass having aviability of 3.15×10¹⁰ CFU/ml was obtained; accumulated zinc: 63.42mg/g_(DW). SBF32 medium: tryptone 23 g/l; soybean peptone 16 g/l; yeastextract 12 g/l; MgSO₄ 0.25 g/l; K₂HPO₄ 2.5 g/l; ascorbic acid 0.5 g/l;lactose 45 g/l; glycerine 19 g/l.

EXAMPLE 4

71 ml of M17 medium, to which 10 mM zinc sulphate has been added(separately sterilised by filtration) were inoculated with 500 ml ofseed culture liquor from the same M17 medium+10 mM zinc sulphate intowhich Streptococcus thermophilus ST 16 BM (accession number DSM 19526;filing date 13.7.2007) has previously been grown for 24 hours at 42° C.under anaerobic conditions. Fermenter conditions: 150 rpm; 0.5 lair/l/min; temperature: 42° C.; pH adjusted to 4.8 (±0.2) with 10% NaOH;fermentation time: 21 hours. 2.75×10¹⁰ CFU/ml were obtained. The totalzinc accumulated by the cells was 57.2 mg/g_(DW).

EXAMPLE 5

As in Example 4, but two additions of 15 mM Zn²⁺ were made duringfermentation at log12 and log18. Fermentation time: 24 hours. Cellviability at the end of the fermentation: 2.25×10¹⁰ CFU/ml. The totalzinc accumulated by the cells was 91.3 mg/g_(DW).

EXAMPLE 6

300 ml of MRS medium (Merck)+cysteine, sterilised at 120° C. for 30minutes, to which filter-sterilised 10 mM zinc sulphate has been added,were kept in pre-reduction for not less than 24 hours under aerobicenvironment. A Bifidobacterium animalis subsp. lactis BB 1 BM (accessionnumber DSM 17850; filing date Dec. 23, 2005) seed culture liquor,previously grown for 24 hours in the same medium under anaerobicconditions at 37° C., was inoculated into the abovementioned 300 ml at10% (v/v) and grown under anaerobic conditions for 24 hours at 37° C.1×10¹¹ CFU/ml were obtained at the end of the culture. The total zincaccumulated by the cells was 53.32 mg/g_(DW).

EXAMPLE 7

A 15 litre anaerobic fermenter fitted with a shaker plate in place ofthe shaft was prepared with 10 litres of medium and in the sameconditions as in Example 6. Zinc sulphate was adjusted to 15 mM. Thefermenter was inoculated with 10% v/v of a 24 hour Bifidobacteriumanimalis subsp. lactis BB 1 BM (accession number DSM 17850; filing dateDec. 23, 2005) seed culture liquor and incubated at 37° C. for 24 hours.The amount of living cells was 1×10¹¹ CFU/ml. The total zinc accumulatedby the cells was 67.1 mg/g_(DW).

EXAMPLE 8

90 ml of MRS medium containing 0.05% cysteine, to which 10 mM Znsulphate has been added, were sterilised in a 100 ml flask. ABifidobacterium animalis subsp. lactis BB 1 BM seed culture, previouslygrown for 24 hours at 37° C. under anaerobic conditions in the samemedium, was inoculated into the flask in the amount of 10% v/v. Theculture was then left growing for 40 hours at 37° C. under anaerobicconditions. 3.12×10⁹ CFU/ml were obtained at the end of the culture. Thebiomass was collected by centrifugation and treated and analysedaccording to the disclosed method. The total Zn accumulated by the cellswas 54.36 mg/g p.s.

EXAMPLE 9

90 ml of minimal basal medium (in grams per litre) were formulated asfollows: Casamino acids (Difco Laboratories, Sparks, Md.), 15; yeastnitrogen base (Difco Laboratories), 6.7; ascorbic acid, 10; sodiumacetate, 10; (NH₄)₂SO₄, 5; urea, 2; MgSO₄.7H₂O, 0.2; Fe—SO₄.7H₂O, 0.01;MnSO₄.7H₂O, 0.007; NaCl, 0.01; Tween 80, 1; cysteine, 0.5 (pH adjustedto 7.0 and autoclaved for 30 minutes at 110° C.). One of the followingcarbohydrates (glucose, fructo-oligosaccharides, inulin, raffinose,lactose, galacto-oligosaccharides, fructose, galactose orxylo-oligosaccharides) was autoclaved separately and added to the basalmedium in order to achieve a 10 g/l concentration. 10 mM Zn sulphate wasfurther added. A Bifidobacterium animalis subsp. lactis BB 1 BM seedculture, previously grown for 24 hours at 37° C. under anaerobicconditions in the same medium, was inoculated into the flask in theamount of 10% v/v. The culture was then left growing for 40 hours at 37°C. under anaerobic conditions. Biomass concentrations in the range from1.5×10⁸ CFU/ml to 3.2×10⁹ CFU/ml were obtained at the end of theculture. The biomass was collected by centrifugation and treated andanalysed according to the method described. The total zinc accumulatedinto the cells was in the range from 48.12 to 54.37 mg/g p.s.

EXAMPLE 10

2 litres of MRS medium containing 0.05% cysteine and added with 10 mM Znsulphate, were sterilised in a 3.6 litre bioreactor and inoculated at10% with a Bifidobacterium animalis subsp. lactis BB 1 BM culture grownfor 24 hours in the same medium. The bioreactor was sterilised in situand pressurized with nitrogen. The process conditions were: constantnitrogen insufflation at 0.01, 150 rpm, 37° C., pH maintained at 6.2with 0.1 M NaOH. After 48 hours the biomass had a concentration of3.6×10⁹ CFU/ml. The biomass was collected and analysed for the amount ofzinc. The total zinc accumulated into the cells was 53.81 mg/g p.s.

EXAMPLE 11

2 litres of MRS medium containing 0.05% cysteine were sterilised in a3.6 litre bioreactor and inoculated at 10% with a Bifidobacteriumanimalis subsp. lactis BB 1 BM culture grown for 24 hours in the samemedium. The bioreactor was sterilised in situ and pressurized withnitrogen. The process conditions were: constant nitrogen insufflation at0.01, 150 rpm, 37° C., pH maintained at 6.2 with 0.1 M NaOH. After a 24hour growth period, Zn sulphate was added to the culture in order toachieve a 10 mM final concentration. 24 hours from the addition of themetal, the biomass had a concentration of 3.6×10⁹ CFU/ml. The biomasswas collected and analysed for the amount of zinc. The total zincaccumulated into the cells was 53.81 mg/g p.s.

EXAMPLE 12

10 litres of MRS medium containing 0.05% cysteine with an addition of 10mM Zn sulphate, were sterilised in a 3.6 litre bioreactor and inoculatedat 10% with a Bifidobacterium animalis subsp. lactis BB 1 BM culturegrown for 24 hours in the same medium. The bioreactor was sterilised insitu and pressurized with nitrogen. The process conditions were:constant nitrogen insufflation at 0.01, 150 rpm, 37° C., pH maintainedat 6.2 with 0.1 M NaOH. After the culture had finished acidifying, asindicated by the termination of NaOH attraction, the culture wassupplied in fed-batch mode with a 30% glucose and 10 mM Zn sulphatesolution. After having reached a 15 litre volume, the biomass had aconcentration of 1.2×10¹⁰ CFU/ml and was collected and analysed for theamount of zinc. The total zinc accumulated into the cells was 52.15 mg/gp.s.

1. A method of preparing a zinc-enriched biomass, the method comprising:(i) culturing micro-organisms selected from the group consisting ofBifidobacterium animalis, Streptococcus thermophilus, and combinationsthereof, in a nutrient culture medium comprising a zinc salt, to obtainzinc-enriched micro-organisms; and (ii) separating the zinc-enrichedmicro-organisms from the culture medium.
 2. The method according toclaim 1, wherein the micro-organisms separated from the culture mediumare subjected to freeze-drying, drying, micro-encapsulation and/orfreezing.
 3. The method according to claim 1, wherein themicro-organisms are selected from the group consisting of Streptococcusthermophilus ST 16 BM, deposited under the Budapest Treaty at the DSMZ,Braunschweig, Germany, under the accession number DSM 19526 on Jul. 13,2007, and Bifidobacterium animalis subsp. lactis BB 1 BM, depositedunder the Budapest Treaty at the DSMZ, Braunschweig, Germany, under theaccession number DSM 17850 on Dec. 23,
 2005. 4. The method according toclaim 1, wherein the zinc salt is zinc sulphate.
 5. The method accordingto claim 4, wherein the nutrient culture medium comprises a zincsulphate amount in the range from 5 to 50 mM.
 6. The method according toclaim 1, wherein the culture medium is a liquid medium.
 7. The methodaccording to claim 1, wherein the culture medium comprises theconventional nutrients for the growth of said micro-organisms, selectedfrom the group consisting of inorganic salts, carbon, nitrogen,vitamins, and micro-nutrient sources, and mixtures thereof.
 8. Themethod according to claim 1, wherein said culture medium has a pH in therange from 2.5 to 8.0.
 9. The method according to claim 1, wherein saidmicro-organisms are cultured in said culture medium at a temperature inthe range from 25° C. to 48° C.
 10. The method according to claim 1,wherein said micro-organisms are cultured in said culture medium for 6to 40 hours.
 11. The method according to claim 1, wherein saidmicro-organisms are separated from the culture medium by centrifugationor micro-filtration.
 12. A zinc-enriched biomass obtainable by themethod according to claim
 1. 13. The biomass according to claim 12,comprising micro-organisms selected from the group consisting ofStreptococcus thermophilus ST 16 BM, deposited under the Budapest Treatyat the DSMZ, Braunschweig, Germany, under the accession number DSM 19526on Jul. 13, 2007, and Bifidobacterium animalis subsp. lactis BB 1 BM,deposited under the Budapest Treaty at the DSMZ, Braunschweig, Germany,with the accession number DSM 17850 on Dec. 23, 2005, and combinationsthereof.
 14. The biomass according to claim 12, the biomass comprisingliving micro-organisms.
 15. A pro-biotic agent comprising the biomassaccording to claim
 14. 16. A method for manufacturing a compositionhaving pro-biotic activity, the method comprising: combining the biomassof claim 14 with suitable vehicles and/or excipients.
 17. The method ofclaim 16, wherein said composition having pro-biotic activity isselected from the group consisting of food supplements, dietaryproducts, functional foodstuffs, nutraceutic products, and foodpreparations.
 18. The biomass according to claim 12, comprising deadmicro-organisms.
 19. A method for manufacturing a cosmetic product or afood product, the method comprising: combining the biomass of claim 18with suitable vehicles and/or excipients
 20. A composition havingpro-biotic activity comprising the zinc-enriched living biomassaccording to claim 14, in combination with suitable vehicles and/orexcipients.
 21. The composition according to claim 20 with a bacterialload of at least 10⁹ CFU/g.
 22. A food preparation, food supplement,dietary product, functional food, nutraceutic product, or cosmeceuticproduct, comprising the composition according to claim
 20. 23. Acosmetic or food product comprising the biomass according to claim 18.24. A micro-organism selected from the Streptococcus thermophilus ST 16BM strain, deposited under the Budapest Treaty at the DSMZ,Braunschweig, Germany, under the accession number DSM 19526 on Jul. 13,2007, and the Bifidobacterium animalis subsp. lactis BB 1 BM strain,deposited under the Budapest Treaty at the DSMZ, Braunschweig, Germany,under the accession number DSM 17850 on Dec. 23,
 2005. 25. The methodaccording to claim 4, wherein the nutrient culture medium comprises azinc sulphate amount in the range from 10 to 40 mM.
 26. The methodaccording to claim 1, wherein said culture medium has a pH in the rangefrom 3.5 to 7.5.
 27. The method according to claim 1, wherein saidmicro-organisms are cultured in said culture medium at a temperature inthe range from 35° C. to 45° C.
 28. The method according to claim 1,wherein said micro-organisms are cultured in said culture medium for 8to 36 hours.